Antibiotic purification and complex compounds



United States Patent Edwin W. Blase, Huntington Station, N. Y., assignorto Chas. Pfizer & Co., Inc, Brooklyn, N. Y., a corporation of DelawareNo Drawing. Application May 18, 195s Serial No. 585,627

4 Claims. (Cl. 260-295) This invention is concerned with a method forthe purification of the broad-spectrum antibiotic oxytetracycline andwith certain complex. compounds of this antibiotic.

Oxytetracyclinc, which is available commercially under the registeredtrademark Terramycin, is a broadspectrum antibiotic of considerablevalue in therapy. The antibiotic is referred to herein as anoxytetracycline antibiotic. This term is meant to include not only theamphoteric compound, but also salts of this compound with metals such asthe alkali metals or alkaline earth metals, or :with other polyvalentmetals and With acids such as the mineral acids. 7

Oxytetracycline is normally produced by for instance with the use of theorganism Streptomyces rimosus. 'In this case, the antibiotic is obtainedin the fermentation as a dilute aqueous solution, highly coutaminatedwith a variety of organic and inorganic impurities from which it must beseparated in order that it may be used as a pharmaceutical. .Even whenthe antibiotic is to be used as an industrial bactericide or for animalfeed enrichment, it is often desirable to bring about at least a partialpurification and to isolate the antibiotic in the form of a solidcompound more readily blended with other materials such as animal feeds.

The present application is a continuation-in-part based on U. S. SerialNo. 412,647, filed February 24, 1954, and now abandoned, which in turnis based on an earlier filed, now abandoned application, Serial No.194,566, filed on November 7, 1950, by Edwin W. Blase.

It has been found that oxytetracycline may be precipitated from impuredilute aqueous solutions thereof in the form of a product of limitedaqueous solubility by treating the impure aqueous solution of theantibiotic with certain organic bases. The organic bases used in theprecipitation of the :antibiotic must have certain properties in orderthat they may be used for the precipitation. In

general, these must be of sufficiently high molecular weight so that aprecipitate of low solubility is formed and yet the organic bases mustnot be of too high a molecu- 'lar weight or their solubility in water isso limited that inefficient precipitation, if any, is obtained. Thepresence of polyvalent metallic ions in the aqueous solution from whichthe antibiotic is precipitated seems to be essential for the mostsuccessful operation of the process. mally fermentation broths containan appreciable amount of such polyvalent metals, generally in the formof cal-.

cium, magnesium or other non-toxic polyvalent ions. If insufiicient ispresent, it can readily be added tothe solution to assure the mostcomplete precipitation of the antibiotic by means of the organic base.

A variety of different organic bases, including organic amines andquaternary ammonium compounds may. be used in the present process toprepare the new basic complexes of oxytetracycline. In general, anorganic base having a reasonable solubility in water and yet ofsufficiently high molecular weight is preferred so that ametallo-organic base complex is formed with the oxyfermentation,

Nor-

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Patented Feb. .10, 1959 tetracycline which has a sufficiently lowsolubility in water to make possible the recovery of the antibiotic fromtypical fermentation broths. These compounds, of course, vary in theirquantitative efficiency, some of them removing only a minor part of themicrobiological activity from higher potency broths, others removing ormore of the potency. The following classes of organic bases have beenfound to be particularly effective in the process. It should be realizedthat a number of organic bases having similar structures may be foundwhich work more or less efficiently in thesame process.

(a) Primary alkyl amines having from 6 to 12 carbon atoms in a straightor branched chain. This includes such compounds as n-octylamine,n-hexyamine, I-methylheptylamine, decylamine, and so forth.

(5) Secondary alkyl amines in which two alkyl groups having between 6and 9 carbon atoms are attached to the nitrogen. These chains may bestraight or branched and the alkyl groups may be identical or differwithin the indicated limitation. Examples of this type of compound aredi-n-hexylamine, di-n-octylamine, n-hexyl-noctylamine,di-Z-ethylhexylamine, di-heptylamine.

(c) l-short chain alkyl (or l-hydroxy substituted short chainalk'yl)-2-long chain alkyl-irnidazolines In these compounds, the shortchain alkyl group has between 1 and 4 carbon atoms andan hydroxyl groupmay be substituted on any of these carbon atoms. The alkyl group on the2-position has between 7 and 17 carbon atoms. A number of thesecompounds are commercially available and they include such compounds asAmine C (Geigy Chemical Corporation), Amine O (Geigy), Amine S (Geigy),Amine 121 (a 1-fi-hydroxyethylimidazoline prepared from the mixture ofamines derived commercially from coconut oil, such that'the 2-positionof the imidazoline is substituted with a mixture of alkyl groups of fromabout 7 to 17 carbon atoms).

(d) Primary phenyl alkyl amines in which the alkyl group has 1 to 3carbon atoms such as benzylamine, phenethylamine, ot-methylbenzylamine,\ot-ethylbenzylamine and so forth.

(e) Long chain alkyl-tri-short chain alkyl ammonium halides wherein thelong chain has 8 to 18 carbon atoms, the short chains have 1 to 3 carbonatoms and the halide is chloride, bromide, or iodide. A variety of thesecompounds are commercially available and useful in this process, such asoctadecyl-trimethylammonium chloride, decyl-triethylammonium chloride,dodecyl-trimethylammonium bromide, cetyl-trimethylammonium chloride,mixtures of trimethylammonium salts of the mixture of amines derived bya commercially operated process from soybean fatty acids, from coconutoil fatty acids and from other such sources.

(f) Long chain alkyl-di-short chain alkyl-benzylammonium halides whereinthe long chain alkyl group has from 8 to 18 carbon atoms, the shortchain alkyl groups have from 1 to 3 carbon atoms eachand the halide iseither chloride, bromide, or iodide. A number of these compounds arecommercially available or may be easily prepared such ascetyl-dimethylbenzyl-ammonium-chloride, Onyx BTC 824 (a mixedalkyl-dimethylbenzyl-ammouium chloride), Onyx BTC 927 (a mixedalkyl-dimethyl-(dimethylbenzyl)-ammonium chloride),dodecyldiethyl-benzylammonium bromide.

(g) (Medium chain alkyl) benzyl-tri-short chain alkyl ammonium halideswherein the medium chain attached to the benzyl group (at the ortho,meta or para position) has 4 to 8 carbon atoms and the short chain alkylgroups have 1 to 3 carbon atoms each. Compounds of this nature arecommercially available and include(tertiarybutyl)benzyl-trimethylammonium chloride,(tertiary-octripropylammonium bromide, etc.

(h) Di-1nedium chain alkyl-di-short chain alkyl antmonium halideswherein the medium chain alkyl groups have from 6 to 18 carbon atoms,and the short chain alkyl groupss have 1 to 3 carbon atoms. The halideis bromide, chloride, or iodide. Examples include didodecenyl dimethylammonium chloride, dioctyl dipropyl ammonium bromide, didecyl diethylammoniu iodide, di- 2-et-hylhexyl diethyl amonium chloride, etc.

i (i) l-short chain alkyl-Z-long chain alkyl-3-short chain alkyl (orbenzyl) imidazolinium halides wherein the short chain alkyl grouphas-from 1 to 4 carbon atoms and may be substituted with an hydroxylgroup on any one of these carbon atoms. The long chain alkyl group hasfrom 7 to 17 carbon atoms. A number of these compounds are commerciallyavailable, being formed by quaternization of the correspondingimidazoline compounds with an alkyl halide or an aralkylhalide. Examplesof this type of compound include Quaternary l2lQb which is an imid-'azolinium chloride with a ,B-hydroxyethyl substituent at the l-positionand a benzyl group at the 3-position prepared from the imidazolineformed from the amines derived from coconut oil fatty acids andQuaternary 12lQc, a compound of similar structure with a chlorobutylgroup at the 3-position.

(j) (Medium chain alkyl)phenoxyethoxyethyl di-short chain alkyl benzylammonium halides wherein the medium chain alkyl is branched or straight,has 6 to '12 carbons, and it is attached at the ortho, meta or paraposition to the phenoxy group, the short chain alkyls have 1 to 3carbons each, and the halide is chloride, bromide or iodide. Examples ofsuch compounds are p-(di-tertiary butyl)phenoxyethoxyethyl dimethylbenzyl ammonium chloride, p-hexyl-phenoxyethoxyethyl diethyl benzylammonium bromide, o-decyl-phenoxyethoxyethyl dipropyl benzyl ammoniumiodide, etc.

(k) N,N-di((medium chain alkyl benzyl) dimethyl ammonium acetyl)ethylenediamine dihalide wherein the medium chain alkyl substituted onthe ring of the benzyl group is of 6 to 12 carbons and the halide ischloride, bromide, or iodide. Examples include the p'di-tertiary butylbenzyl compounds, the o-hexyl compounds, the pdodecyl compounds, etc.

(I) N-(long chain alkanoyl ethanolaminoformylmethyl)pyridinium halides,wherein thelong chain alkanoyl group is a carboxylicacid group having 8to 18 carbons and the halide is bromide, chloride or iodide. Examplesinclude N- (lauroyl ethanolaminoformylmethyl pyridinium chloride (EmcolE 607), N-(stearyl-ethanolaminoformylmethyl)pyridinium bromide, N(tetradecanoyl ethanolaminoformylmethyl)pyridinium iodide.

One particularly useful basic organic precipitant for oxytetracyclinefermentation broths is the material known as Arquad C. This is availablecommercially as a 50% by weight solution in isopropanol or a 33% aqueoussolution. It consists of a technical mixture comprising about 50%dodecyl-trimethylammonium chloride, together with lesser amounts of'octadecyl-, tetradecyl-, hexadecyl-, octadecenyl-, octylanddecyl-trimethylammonium chlorides. The small amount of isopropanolintroduced when that solution is used does not interfere seriously withthe efficiency of precipitation of the oxytetracycline antibioticcomplexes. As little as 0.5% by volume of the 33% aqueous'solution maybe used quite successfully in the present process and fermentationbroths are generally thereby reduced in potency to less than 100 meg/ml.Decreasing the proportion of reagent tends to leave more of theoxytetracycline still dissolved in solution. It is usually practical toincrease the proportion up to about 2% Excess precipitant may partiallydissolve the precipitate. In general, a proportion of about 1 to aboutgrams on a dry basis of this particular reagent per liter of broth ispreferred. Other precipitants are also effective in approximately thisproportion, although the lower molecular weight compounds are requiredin somewhat smaller amounts and higher molecular weight materials tionis adjusted. The use of pI-Is lower than about 5.5

is impractical while pHs from about 6 to about 10 give excellentresults. Since the stability of oxytetracycline decreases with higherpHs, a pH of approximately 8.0 to 9.5 is most effective. The proceduregenerally employed in recovering oxytetracycline from a microorganismfermentation broth is to filter the mycelium from the acidified broth,add the desired organic base precipitant (which may be in the form ofone of the above amines or as a water soluble salt of such amine, e. g.the hydrochloride, hydrobromide, acetate, citrate, tartrate, etc. or oneof the quaternary compounds) and then adjust the pH of the solution Witha soluble alkali, such as caustic soda or potash. Other alkalies such assodium carbonate, potassium carbonate, lithium hydroxide, lime, and soforth may be used for this purpose. It has been observed that the rapidaddition of an alkaline solution tends to precipitate a finely dividedproduct which may be difficult to filter. Slower addition gives a morereadily filterable product.

Precipitates Which are obtained by treatment of oxytetracyclineantibiotic solutions with the various operable organic bases describedabove in the presence of polyvalent metallic ions do not consist ofsimple salts of the antibiotic and the chosen base, but rather arecomplex salts thereof containing various metallic ions which areprecipitated during the process. This may be demonstrated by the factthat no appreciable precipitate results upon treating a dilute aqueoussolution of pure oxytetracycline antibiotic, comparable in concentrationto a conventional oxytetracycline antibiotic fermentation broth, with anamine or quaternary ammonium salt which is known to precipitate theoxytetracycline from fermentation broths. However, the addition of smallamounts of bior trivalent metallic ions to such a pure oxytetracyclinesolution causes ready precipitation of complex antibiotic compounds.These contain, in addition to the antibiotic, the organic base and oneor more polyvalent metallic ions. The latter are normally present infermentation broths. For instance, a typical precipitate obtained froman oxytetracycline fermentation broth treated with Arquad C was analyzedand found to contain approximately 51% oxytetracycline, 19% of thequaternary ammonium compound and 7% ash. The metallic components of theash were approximately 45% magnesium and about 45% calcium. Since mediaused in the preparation of oxytetracycline by fermentation vary somewhatin their metallic ion composition, the resulting precipitate obtained bythe present process will also vary in its metallic ion content. Normallyoxytetracycline antibiotic fermentation media contain sufiicientpolyvalent metallic ions to assure the maximum precipitation of theantibiotic when a suitable organic base is added to the filtered brothand the pH is adjusted to a suitable basic value. In fact, the brothsoften contain more metallic constituents than is required, inwhich casethe excess may be preliminarily removed by any desired means; forinstance, by the sequestration method taught in U. S. Patent No. 2,658,078. In this method acidic clarified oxytetracycline broth is treatedwith a sequestering agent for polyvalent metallic ions. Such agentsbeing Weakly ionized hydroxylated organic'acid compounds, like citricacid or gluconic acid,

sodium tetraphosphate and so forth, and amino acids' such asethylenediamine tetraacetic acid and polyaminocarboxylic acid salts suchas the commercially-available product Perma-Kleers. If theoxytetracycline .antibiotic broth should contain insuflicient metallicions, iron, zinc, magnesium, calcium, strontium and the like may beadded to assist the precipitation still further. In fact, any reasonablysoluble polyvalent metallic ion may be used for this purpose. If an"impure aqueous solution of oxytetracycline is obtained from anothersource than fermentation, such polyvalent metals must, of course, beadded in order to carry out the precipitation procedure of the presentinvention. The various organic bases used in the present process varysomewhat in their selectivity as would be expected; that is, theproportion of antibiotic present in the precipitated salts does notremain constant with different precipitants. The composition of theprecipitate will also obviously vary somewhat with the pH used duringthe precipitation.

Although it is possible to use solutions or broths having anoxytetracycline antibiotic potency of 500 mcg./ ml. or lower, those arepreferred which are somewhat higher in potency, that is, at least about1000 mcg./ml., in order that the. product will have suitably highpotency and will be obtained in good yield. The various organic basesvary in their effectiveness of precipitation; for instance, some of thecompounds referred to above may precipitate only a portion of a typicalbroth containing 1000 mcg./ml., others will precipitate 75% or more. Ingeneral, only a fraction of the quaternary ammoniumsalt or other organicbase precipitant is found in the resulting precipitate; that is, anexcess should be used in order to obtain a reasonably complete recoveryof the antibiotic. Somewhat over a three-fold molar excess of thereagent is most successful. This will vary with the individual base andwith the nature of the polyvalent metallic ions present in the solutionused for the precipitation. A minimum of experimentation will establishthe most suitable ratio for a given fermentation broth.

The product which is formed by the precipitation process of the presentinvention, when separated from solution and dried, generally assays fromabout 300 to about 500 meg/mg. of oxytetracycline activity. A one-stepoperation thus serves greatly to concentrateand purify the antibiotic,since the fermentation broth normally contains, on a dry basis,oxytetracycline at an appreciably lower concentration. The complex saltprecipitate may readily be treated to recover pure oxytetracycline orits simple acid salts, or it may be used directly in therapeutic ornutritional fields or as an industrial antibacterial agent. Forinstance, the quaternary ammonium salts of high molecular weight areparticularly strong germicides in their own right showing an activitywhich is greater than could be expected from the oxytetracycline contentand the quaternary ammonium salt content.

The complex metallo-organic salts of oxytetracycline obtained by thepresent process may be treated to regenerate the oxytetracycline contentby dissolving them in lower alcohols and ketones, preferablyafter theremoval ofexcessmoisture by filtration and drying. Methanol is mostsatisfactory, being low in cost and easily recovered. Otherloweralcohols such as ethanol and lower ketones such as acetone arequite useful. An alternative recovery of the anti-biotic involves thetreatment of the organic base complex in a lower aliphatic alcohol withhydrochloric acid or a polyvalent metallic halide soluble in the solventto bring about dissolution of the precipitated product. The solution maythen be clarified, if desired, by means of a decolorizing adsorbent suchas bone black. The addition of further hydrochloric acid to the solutionresults in the separation of a highly purified, generally crystallineacid salt of oxytetracycline.

1 In recovering the antibiotic from the precipitate and preparing acrystalline salt, a methanolic solution containing from about 4 to 8liters of the solvent per kilogram of oxytetracycline activity isgenerally prepared and then treated with concentrated hydrochloric acid.Care must be exercised to avoid precipitation of amphotericoxytetracycline. It is particularly convenient to add the dried organicbase-oxytetracycline antibiotic precipitate to the methanol solventwhile the pH of the solution is continually adjusted from about 2 toabout 2.5 by the addition of concentrated hydrochloric acid. Calcium 6chloride'may be used to replace or partially replace the hydrochloricacid in obtaining a clear solution of the antibiotic. Any small amountof solid impurities may be filtered from the solution. Further additionof con centrated acid, for instance, hydrochloric acid, causes formationof the desired simple antibiotic salt, such as the hydrochloride. Whenhydrochloric acid is used preliminarily to aid dissolution of thecomplex salt, from about 0.4 to about 0.7 ml. of the concentrated acid(36%) is required to dissolve the metallo-organic salt equivalent to onegram of oxytetracycline antibiotic activity, and solutions havingpotencies of about 60,000 to 150,000 mcg. of oxytetracycline per ml. orhigher are thus prepared. This clear solution may then be treated withan adsorbent-like decolorizing carbon to remove a variety ofcontaminating impurities. About 0.5 to about 1.5 grams of carbon isgenerally used per gram of oxytetracycline potency in the solution. Alarger proportion may be used giving a product of greater purity withoutmuch loss due to adsorption of the antibiotic. The adsorbent may beremoved with the assistance of a filteraid and washed with an acidifiedsolvent to remove residual material.

Crystalline oxytetracycline hydrochloride or similar salts of goodquality are prepared from the clarified solution thus obtained. This isaccomplished'by the addition of the salt-forming acid, such asconcentrated hydrochloric acid. The acid salt crystallizes from thesolution. The solution may be concentrated after crystallization, ifdesired, to obtain a more complete crystallization of the product. Ingeneral a ratio of at least about 200 ml. of concentrated hydrochloricacid per kilogram of the antibiotic present in the solution is required.Somewhat larger amounts of acid may improve the yield of crystallineproduct and 0.95 liter of the concentrated hydrochloric acid perkilogram of oxytetracycline is a preferred proportion. However, thiswill vary to some extent with the type of precipitant used and thenature of impurities present in the material. The optimum conditi-onsmay be obtained by the minimum experimentation by a skilled individual.The use of more than one liter of acid per kilogram of antibiotic is notrecommended since it may lead to some loss of product. Crystallizationis most practically conducted at room temperature, although a lowertemperature may give a slightly better yield of product. A highertemperature may induce some loss of product.

In addition to regenerating the oxytetracycline by directcrystallization of its acid salts from the metallo organic compounds,regeneration may be effected by dissolving such compounds in an organicsolvent like methanol with the aid of an acid. The presence of a minorproportion of water is not harmfui. Sulfuric acid is most often used forthis purpose. Precipitated impurities such as calcium sulfate areremoved by filtration and the pH is raised to a point within the rangeof from about 5 to about 7. A precipitate containing a high proportionof free amphoteric oxytetracycline is thus ob tained. Small amounts ofpolyvalent metals may be coprecipitated. The presence of a sequesteringagent, such as ethylenediamine tetraacetic acid, assists in thepreparation of amphoteric antibiotic of higher purity. This may beconverted to a crystalline acid salt by conventional means, if desired.Organic solvents may be removed by distillation and replaced by waterbefore filtering. This raises the yield to a certain extent.Alternatively, the product may be filtered and the solvent removed toobtain a second precipitate of somewhat lower potency which may berecycled for further treatment.

In the recovery of pure oxytetracycline antibiotic or its simple acidsalts, such as the hydrochloride, from solutions of the metallo-organiccomplexes, the organic base used as precipitant and some of theantibiotic itself, are left in the mother liquor and wastes. It may bedesirable to reclaim and reuse such materials; for instance, if the,mother liquor is recycled and'added to the next batch of oxytetracyclineantibiotic solution or broth to be treated according to the presentinvention, the amount of organic base required for precipitation may besubstantially reduced without appreciably lowering the yield ofoxytetracycline precipitated. Part or all of the solvent may first beremoved from the mother liquor before its addition to the next batch.This-is not essential. Certain colored impurities may be returned in themother liquor and give a somewhat darker oxytetracycline antibiotic saltproduct. If the recycled operation is repeated too many times, thisdarkening of the product may be troublesome and the yield may decrease.However, recycling the mother liquor a few times is feasible and doesnot harm the quality of the product.

The process of the present invention has many advantages. It uses cheap,readily available reagents of low toxicity, a crystalline salt of highquality is obtained from highly dilute and/ or very crudeoxytetracycline antibiotic solutions, and the procedure involves aminimum number of steps and produces good yields of the antibiotic.

The following examples are given to illustrate this invention and arenot intended to limit its scope.

Example I A volume of acidified filtered oxytetracycline brothcontaining 25.3 grams of the antibiotic was treated with grams of citricacid per liter of solution to sequester a portion of the polyvalentmetallic ions therein. Five ml. of 33% aqueous Arquad C per liter ofbroth were then added thereto, and the pH of the solution was adjustedto 9 by introducing sodium hydroxide solution over a period of one hour.A complex precipitate containing oxytetracycline, mixed alkyl trimethylammonium bases and various metals resulted and was filtered using afilteraid. The residual filtrate was assayed and found to contain 0.83gram of oxytetracycline activity. The filter cake was dried at 50 C.under vacuum and then milled. The resulting finely divided solid wasextracted with sufficient methanol so that a solution containing 136 mg.of oxytetracycline per ml. was obtained. This solution was filtered andconcentrated hydrochloric acid was gradually added thereto with constantagitation. Crystalline oxytetracycline hydrochloride separated. It wasfiltered, washed with acidified methanol and dried. A total of 14.2grams of purified, crystalline oxytetracycline hydrochloride was thusobtained by a simple procedure.

Example 11 Filtered oxytetracycline broth containing 25.3 grams ofoxytetracycline activity was treated with 0.2% of Arquad C (33% aqueoussolution). To this solution was added the mother liquor obtained duringthe crystallization of the oxytetracycline hydrochloride prepared inExample I. This recycled liquor contained 6.6 grams of oxytetracyclineactivity. The mixed solution was adjusted to pH 9 by the slow additionof sodium hydroxide solution, and the resulting precipitate was filteredand dried. The filtrate contained 1.08 grams of the antibiotic. Theprecipitate comprised complex metalloquaternary ammonium salts ofoxytetracycline, wherein the total antibiotic content was 29.8 grams.This product was dissolved in methanol and concentrated hydrochloricacid was simultaneously added at the rate of 0.48 ml. of acid per gramof oxytetracycline activity. A solution contain,- ing 62.5 mg. ofantibiotic per ml. was obtained. This was treated with 0.99 gram of acommercially available activated carbon per gram of oxytetracyclineactivity. After stirring for /2 hour, the carbon was filtered with theassistance of a filteraid. To the clarified solution was added 0.19 gramof anhydrous calcium chloride per gram of oxytetracycline, in order toprevent the premature crystallization of amphoteric oxytetracycline andto assure formation of oxytetracycline hydrochloride of high quality. Tothe clear solution was added 0.60 ml. of concentrated hydrochloric acidper gram of antibiotic present. This resulted in the formation ofcrystalline oxytetracycline hydrochloride. The precipitated crystalswere filtered, carefully washed with acidified methanol and then withmethanol and dried under vacuum at 50 C. A total of 15.75 grams of thecrystalline acid salt was obtained. The mother liquor from thecrystallization contained 19.3 mg. of antibiotic per ml., a total of8.56 grams of the antibiotic. More of the antibiotic was recoverable bya second extraction of the filter cakes and of the decolorizing carbon.

Example III Volume, Potency of Potency of Reagent m1. Filtrate, Driedmeg/ml. Product,

meg/mg.

Arquad C (33% in water) 2. 5 45 505 Arquad 12 (33% in water) 2. 5 40 480Arquad 12 (50% in IPO) 1.65 40 515 Arquad 18 (50% in IPO) 1. 65 50 445It will be noted that changes in the nature of the organic base used donot appreciably alter the effective removal of the antibiotic from thebroth or the quality of the product.

Example IV A further group of quaternary ammonium salts and certainamines have been demonstrated to be very effective in the precipitationof oxytetracycline from clarified fermentation broth.

Clarified oxytetracycline fermentation broth was'treated with 5 grams ofcitric acid per liter of broth and to one liter portions of this treatedbroth was added 5.0 gram portions (5.0 ml. of the liquids) of thereagents listed below. In each case the solution was then adjusted bythe addition of dilute sodium hydroxide to a pH from about 8.5 to about9.0. The precipitated oxytetracyclinecontaining products were filteredusing a small proportion of a filteraid. The filtrates from each of theprecipitates were assayed for residual antibiotic by means of a standardbiological method. In the following table is reported the reagents undertheir trade names and generic names, as well as the per cent ofoxytetracycline removed from the fermentation broth.

p-Diisobutyl phenoxy ethoxy ethyl dlmeth l benzyl ammonium chloride 96Cetyl dimethyl benzyl ammonium chlorld 98 Decylamine 93 N onylamine. 78Octylamine A high molecular weight, heterocycllc, alkyl substitutedtertiary amine (Amine-O 96 A high molecular weight, heterocyclic, alkylsubstituted teritary amine (Amine-0*) 88 *Irade-name of the commerciallyavailable product.

9 Example V The process described in Example IV was repeated uti lizingas the precipitating agent di-n-hexylamine. A substantial amount of theantibiotic was precipitated from the fermentation broth by the reagent.

Example VI The process described in Example IV was repeated utilizing asthe precipitating agent benzylamine. Part of the antibiotic activity wasremoved from the broth by the precipitant.

Example VII The process described in Example IV was repeated utilizingas the precipitating agent dodecyl-diethyl-benzylammonium bromide. Thisresulted in the precipitation of a major part of the antibiotic from thefermentation broth.

Example VIII The process described in Example IV was repeated utilizingas the precipitating agent didodecenyl dimethyl ammonium chloride. Amajor part of the antibiotic separated from the solution as a complexwith the organic base and polyvalent metal ions in the broth.

Example IX The process described in Example IV was repeated utilizing asthe precipitating agent l-fi-hydroxyethyl-Z- (C to Calkyl)-3-benzyl-imidazolinium chloride (Qua ternar'y-l21Qb). Asubstantial part of the antibiotic activity was precipitated.

Example X The process described in Example IV was repeated utilizing asthe precipitating agent p-(di-tertiary-butyl) phenoxyethoxyethyldimethyl-benzyl ammonium chloride. A major proportion of theoxytetracycline was separated from solution as a precipitate containingthe organic base and calcium and magnesium ions.

Example XII The process described in Example IV was repeated utilizingas the precipitating agent Experimental Quaternary B44 of Rohm & Haas(N,N-di-(medium chain alkyl benzyl) dimethyl ammonium acetyl)ethylenediamine dichloride.

Example XIII The process described in Example IV was repeated utilizingas the precipitating agent l-fl-hydroxyethyl-Z- pentadecyl imidazoline.Part of the antibiotic was precipitated as a complex with the organicbase and polyvalent metal ions.

Example XIV The process described in Example IV was repeated utilizingas a precipitant Emcol E 607. A major part of the oxytetracycline in thebroth was precipitated as a complex.

What is claimed is:

1. A process for the recovery of oxytetracycline from an aqueoussolution thereof obtained from a fcrmenta tion broth, which comprisestreating said aqueous solution in the presence of polyvalent metal ionsat a pH of at least about 5.5 and up to about with at least an excess ofone organic base selected from the group consisting of (a) primary alkylamines having from 6 to 12 carbon atoms; (b) secondary alkyl amineswherein two alkyl groups having between 6 and 9 carbon atoms each areattached to the nitrogen; (c) l-short chain alkyl-Z-long chain alkylimidazolines wherein the short chain alkyl group has between 1 and 4carbon atoms and the long chain alkyl has between 7 and 17 carbon atoms;(d) l-short chain hydroxyalkyl-Z-long chain alkyl imidazolines whereinthe short chain hydroxyalkyl has between 1 and 4 carbon atoms and thelong chain alkyl has between 7 and 17. carbon atoms; (e) primaryphenylalkyl amines wherein the alkyl group has between 1 and 3 carbonatoms; (f) long chain alkyltri-short chain alkyl ammonium halideswherein the long chain alkyl has be tween 8 and 18 carbon atoms and theshort chain alkyl has between 1 and 3 carbon atoms and the halide ischosen from the group consisting of chloride, bromide, and iodide; (g)long chain alkyl di-short chain alkyl benzyl ammonium halides whereinthe long chain alkyl group has between 8 and 18 carbon atoms, the shortchain allcyl group has between 1 and 3 carbon atoms and the halide ischosen from the group consisting of chloride, bromide, and iodide; (11)medium chain alkyl benzyl tri-short chain alkyl ammonium halides whereinthe medium chain is attached to the benzyl group and has between 4 and 8carbon atoms and the short chain alkyl group has between 1 and 3 carbonatoms; (i) iii-medium chain alkyl-di-short chain alkyl ammonium halideswhere in the medium chain alkyl group has between 6 and 18 carbon atomsand the short chain alkyl group has between 1 and 3 carbon atoms and thehalide is chosen from the group consisting of bromide, chloride andiodide; (j) l-short chain alkyl-Z-long chain alkyl-3-short chain alkylimidazoliniurn halides wherein the short chain alltyl group has from 1to 4 carbon atoms, the long chain alkyl group has from 7 to 17 carbonatoms, and the halide is chosen from the group consisting of chloride,bromide and iodide; (k) l-short chain alkyl-Z-long chain alkyl-3-benzylimidazoliniurn halides wherein the short chain alkyl group has from 1 to4 carbon atoms and the long chain alkyl group has from 7 to 17 carbonatoms; (l) l-short chain hydroxyalkyl-Z-long chain allryl-Zl-short chainalkyl imidazoliniurn halides wherein the short chain hydroxyalkyl groupshave from i to 4 carbon atoms, the long chain alkyl. group has from 7 to17 carbon atoms, and the halide is chosen from the group consisting ofchloride, bromide, and iodide; (m) (medium chain alkyl)phenoxyethoxyethyl di-short chain alkylbenzylammonb um halides whereinthe medium chain alkyl group has from. 6 to 12 carbon atoms, the shortchain alkyls have between 1 and 3 carbon atoms each, and the halide ischosen from the group consisting of chloride, bromide, and iodide; (rt)N,N-di-((medium chain alkylbenzyl) dimethyl ammonium acetyl)ethylenediamine dihalides wherein the medium chain alkyl group has from6 to 12 carbon atoms and the halide is chosen from the group consistingof chloride, bromide, and iodide; (0) N-(long chain alkanoylethanolaminoformylrnethyl)pyridinium halides wherein the long chainalkanoyl group has from 8 to 18 carbon atoms and the halide is chosenfrom the group consisting of chloride, bromide and iodide; and

recovering the metal ion-organic base-oxytetracycline antibiotic complexthereby precipitated.

2. A process as claimed in claim 1 wherein the aqueous oxytetracyclinesolution is a fermentation broth.

3. A process as claimed in claim 1 wherein the recovered metalion-organic base-oxytetracycline antibiotic precipitate is treated withdilute acid at a pH below about 5, the antibiotic solution is filtered,and the pH of the filtered solution is adjusted to a value substantiallybetween 5 and 7 and the precipitated oxytetraeycline is recovered.

4. The product produced by the process claimed in claim 1.

References Cited in the file of this patent FOREIGN PATENTS 506,950Belgium Nov. 30, 1951

1. A PROCESS FOR THE RECOVERY OF OXYTETRACYCLINE FROM AN AQUEOUS SOLUTION THEREOF OBTAINED FROM A FERMENTATION BROTH, WHICH COMPRISES TREATING SAID AQUEOUS SOLUTION IN THE PRESENCE OF POLYVALENT METAL IONS AT A PH OF AT LEAST ABOUT 5.5 AND UP TO ABOUT 10 WITH AT LEAST AN EXCESS OF ONE ORGANIC BASE SELECTED FROM THE GROUP CONSISTING OF (A) PRIMARY ALKYL AMINES HAVING FROM 6 TO 12 CARBON ATOMS; (B) SECONDARY ALKYL AMINES WHEREIN TWO ALKYL GROUPS HAVING BETWEEN 6 AAND 9 CARBON ATOMS EACH ARE ATTACHED TO THE NITROGEN; (C) 1-SHORT CHAIN ALKYL-2-LONG CHAIN ALKYL IMIDAZOLINES WHEREIN THE SHORT CHAIN ALKYL GROUP HAS BETWEEN 1 AND 4 CARBON ATOMS; THE LONG CHAIN ALKYL HAS BETWEEN 7 AND 17 CARBON ATOMS; (D) 1-SHORT CHAIN HYDROXYALKYL-2-LONG CHAIN ALKYL IMIDAZOLINES WHEREIN THE SHORT CHAIN HYDROXYALKYL HAS BETWEEN 1 AND 4 CARBON ATOMS AND THE LONG CHAIN ALKYL HAS BETWEEN 7 AND 17 CARBON ATOMS; (E) PRIMARY PHENYLALKYL AMINES WHEREIN THE ALKYL GROUP HAS BETWEEN I AND 3 CARBON ATOMS; (F) LONG CHAIN SLKYL TRI-SHORT CHAIN ALKYL AMMONIUM HALIDES WHEREIN THE LONG CHAIN ALKYL HAS BETWEEN 8 AND 18 CARBON ATOMS AND THE SHORT CHAIN ALKYL HAS BETWEEN 1 AND 3 CARBON ATOMS AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE, AND IODIDE; (G) LONG CHAIN ALKYL DI-SHORT CHAIN ALKYL BENZYL AMMONIUM HALDIES WHEREIN THE LONG CHAIN ALKYL GROUP HAS BETWEEN 8 AND 18 CARBON ATOMS, THE SHORT CHAIN ALKYL GROUP HAS BETWEEN 1 AND 3 CARBON ATOMS AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE, AND IODIDE; (H) MEDIUM CHAIN ALKYL BENZYL TRI-SHORT CHAIN ALKYL AMMONIUM HALIDES WHEREIN THE MEDIUM CHAIN IS ATTACHED TO THE BENZYL GROUP AND HAS BETWEEN 4 AND 8 CARBON ATOMS AND THE SHORT CHAIN ALKYL GROUP HAS BETWEEN 1 AND 3 CARBON ATOMS; (I) DI-MEDIUM CHAIN ALKYL-DI-SHORT CHAIN CHAIN ALKYL AMMONIUM HALIDES WHEREIN THE MEDIUM CHAIN ALKYL GROUP HAS BETWEEN 6 AND 18 CARBON ATOMS AND THE SHORT CHAIN ALKYL GROUP HAS BETWEEN 1 AND 3 CARBON ATOMS AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF BROMIDE, CHLORIDE AND IODIE; (J) 1-SHORT CHAIN ALKYTL-2-LONG CHAIN ALKYL-3-SHORT CHAIN ALKYL IMIDAZOLINIUM HALIDES WHEREIN THE SHORT CHAIN ALKYL GROUP HAS FROM 1 TO 4 CARBON ATOMS, THE LONG CHAIN ALKYL GROUP HAS FROM 7 TO 17 CARBON ATOMS, AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE AND IODIDE; (K) 1-SHORT CHAIN AALKYL-2-LONG CHAIN ALKYL-3-BENZYL IMIDAZOLINIUM HALIDES WHEREIN THE SHORT CHAIN ALKYL GROUP HAS FROM 1 TO 4 CARBON ATOMS AND THE LONG CHAIN ALKYL GROUP HAS FROM 7 TO 17 CARBON ATOMS; (L) 1-SHORT CHAIN HYDROXYALKYL-2-LONG CHAIN ALKYL-3-SHORT CHAIN ALKYL IMIDAZOLINIUM HALIDES WHEREIN THE SHORT CHAIN HYDROXYALKYL GROUPS HAVE FROM 1 TO 4 CARBON ATOMS, THE LONG CHAIN ALKYL GROUP HAS FROM 7 TO 17 CARBONS ATOMS. AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE, AND IODIDE; (M) (MEDIUM CHAIN ALKYL) PHENOXYETHOXYETHYL DI-SHORT CHAIN ALKYLBENZYLAMMONUUM HALIDES WHEREIN THE MEDIUM CHAIN ALKYL GROUP HAS FROM 6 TO 12 CARBON ATOMS, THE SHORT CHAIN ALKYLS HAVE BETWEEN 1 AND 3 CARBON ATOMS EACH, AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE, AND IODIDE; (N) N,N''-DI-((MEDIUM CHAIN ALKYLBENZYL) DIMETHYL AMMONIUM ACETYL) ETHYLENEDIAMINE DIHALIDES WHEREIN THE MEDIUM CHAIN ALKYL GROUP HAS FROM 6 TO 12 CARBON ATOMS AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE, AND IODIDE; (O) N-(LONG CHAIN ALKANOYL ETHANOLAMINOFORMYLMETHYL) PYRIDINIUM HALIDES WHEREIN THE LONG CHAIN ALKANOYL GROUP HAS FROM 8 TO 18 CARBON ATOMS AND THE HALIDE IS CHOSEN FROM THE GROUP CONSISTING OF CHLORIDE, BROMIDE AND IODIDE; AND RECOVERING THE METAL ION-ORGANIC BASE-OXYTETRACYCLINE ANTIBIOTIC COMPLEX THEREBY PRECIPITATED. 